The Ga1 locus of the genus Zea is associated with novel genome structures derived from multiple, independent nonhomologous recombination events.

The Ga1 locus controls cross-incompatibility between field corn and popcorn. The Ga1-S haplotype contains 2 types of pectin methylesterase (PME) genes, ZmPme3 and several copies of ZmGa1P that are expressed in silk and pollen, respectively. The ga1 haplotype contains nonfunctional tandem repeat sequences related to ZmPme3 and ZmGa1P. This haplotype can cross-pollinate freely and is widely present in field corn. The primary objective of this study is to characterize the repeat sequences from a diverse collection of maize and teosinte lines and use this information to understand the evolution of the Ga1 locus. First, we characterized the complexity of the Ga1 genome region in high-quality maize genome assemblies that led to their categorization into 5 groups based on the number and type of PME-like sequences found at this region. Second, we studied duplication events that led to the ga1 and Ga1-S repeats using maximum likelihood phylogenetic reconstruction. Divergence estimates of the ga1 haplotype suggest that the duplication events occurred more than 600 KYA whereas those in Ga1-S occurred at 3 time points, i.e. >600, ∼260, and ∼100 KYA. These estimates suggest that the ga1 and Ga1-S tandem duplication events occurred independently. Finally, analysis of ZmPme3 and ZmGa1P homologs in Zea and Tripsacum genomes suggests that ga1 and Ga1-S repeats originated from an ancestral pair of PME genes that duplicated and diverged through 2 evolutionary branches prior to the domestication of maize.

Usefulness of temperate-adapted maize lines developed by doubled haploid and single-seed descent methods.

KEY MESSAGE: Spontaneous haploid genome doubling is not associated with undesirable linkage drag effects. The presence of spontaneous doubling genes allows maximum exploitation of variability from the temperate-adapted BS39 population Tropical non-elite maize (Zea mays L.) germplasm, such as BS39, provides a unique opportunity for broadening the genetic base of U.S. Corn Belt germplasm. In vivo doubled haploid (DH) technology has been used to efficiently exploit non-elite germplasm. It can help to purge deleterious recessive alleles. The objectives of this study were to determine the usefulness of BS39-derived inbred lines using both SSD and DH methods, to determine the impact of spontaneous as compared with artificial haploid genome doubling on genetic variance among BS39-derived DH lines, and to identify SNP markers associated with agronomic traits among BS39 inbreds monitored at testcross level. We developed two sets of inbred lines directly from BS39 by DH and SSD methods, named BS39_DH and BS39_SSD. Additionally, two sets were derived from a cross between BS39 and A427 (SHGD donor) by DH and SSD methods, named BS39 × A427_DH and BS39 × A427_SSD, respectively. Grain yield, moisture, plant height, ear height, stalk lodging, and root lodging were measured to estimate genetic parameters. For genome-wide association analysis, inbred lines were genotyped using genotype-by-sequencing and Diversity Array Technology Sequencing (DArTSeq). Some BS39-derived inbred lines performed better than elite germplasm inbreds and all sets showed significant genetic variance. The presence of spontaneous haploid genome doubling genes did not affect performance of inbred lines. Five SNPs were significant and three of them located within genes related to plant development or abiotic stresses. These results demonstrate the potential of BS39 to add novel alleles to temperate elite germplasm.

Major locus for spontaneous haploid genome doubling detected by a case-control GWAS in exotic maize germplasm.

KEY MESSAGE: A major locus for spontaneous haploid genome doubling was detected by a case-control GWAS in an exotic maize germplasm. The combination of double haploid breeding method with this locus leads to segregation distortion on genomic regions of chromosome five. Temperate maize (Zea mays L.) breeding programs often rely on limited genetic diversity, which can be expanded by incorporating exotic germplasm. The aims of this study were to perform characterization of inbred lines derived from the tropical BS39 population using different breeding methods, to identify genomic regions showing segregation distortion in lines derived by the DH process using spontaneous haploid genome doubling (SHGD), and use case-control association mapping to identify loci controlling SHGD. Four different sets were used: BS39_DH and BS39_SSD were derived from the BS39 population by DH and single-seed descendent (SSD) methods, and BS39 × A427_DH and BS39 × A427_SSD from the cross between BS39 and A427. A total of 663 inbred lines were genotyped. The analyses of gene diversity and genetic differentiation for the DH sets provided evidence of the presence of a SHGD locus near the centromere of chromosome 5. The case-control GWAS for the DH set also pinpointed this locus. Haplotype sharing analysis showed almost 100% exclusive contribution of the A427 genome in the same region on chromosome 5 of BS39 × A427_DH, presumably due to an allele in this region affecting SHGD. This locus enables DH line production in exotic populations without colchicine or other artificial haploid genome doubling.

Impact of Spontaneous Haploid Genome Doubling in Maize Breeding.

Doubled haploid (DH) technology has changed the maize-breeding landscape in recent years. Traditionally, DH production requires the use of chemical doubling agents to induce haploid genome doubling and, subsequently, male fertility. These chemicals can be harmful to humans and the plants themselves, and typically result in a doubling rate of 10%-30%. Spontaneous genome doubling and male fertility of maize haploids, without using chemical doubling agents, have been observed to a limited extent, for nearly 70 years. Rates of spontaneous haploid genome doubling (SHGD) have ranged from less than 5% to greater than 50%. Recently, there has been increased interest to forgo chemical treatment and instead utilize this natural method of doubling. Genetic-mapping studies comprising worldwide germplasm have been conducted. Of particular interest has been the detection of large-effect quantitative trait loci (QTL) affecting SHGD. Having a single large-effect QTL with an additive nature provides flexibility for the method of introgression, such as marker-assisted backcrossing, marker-assisted gene pyramiding, and systematic design. Moreover, it allows implementation of new methodologies, such as haploid-inducer mediated genome editing (HI-edit) and promotion of alleles by genome editing. We believe the use of SHGD can further enhance the impact of DH technology in maize.

Mapping of QTL and identification of candidate genes conferring spontaneous haploid genome doubling in maize (Zea mays L.).

In vivo doubled haploid (DH) technology is widely used in commercial maize (Zea mays L.) breeding. Haploid genome doubling is a critical step in DH breeding. In this study, inbred lines GF1 (0.65), GF3(0.29), and GF5 (0) with high, moderate, and poor spontaneous haploid genome doubling (SHGD), respectively, were selected to develop mapping populations for SHGD. Three QTL, qshgd1, qshgd2, and qshgd3, related to SHGD were identified by selective genotyping. With the exception of qshgd3, the source of haploid genome doubling alleles were derived from GF1. Furthermore, RNA-Seq was conducted to identify putative candidate genes between GF1 and GF5 within the qshgd1 region. A differentially expressed formin-like protein 5 transcript was identified within the qshgd1 region.